RB1 circulating-tumor DNA in the blood of Retinoblastoma patients increases in untreated patients.

PURPOSE: Circulating tumor DNA (ctDNA) in plasma has been identified in many cancers, including retinoblastoma at diagnosis. We have previously shown that with treatment (enucleation or ophthalmic artery chemosurgery (OAC)) all ctDNA disappears; and if there is persistent plasma ctDNA after treatment metastases develop. The purpose of this study was to determine how the ctDNA RB1 variant allele frequency (VAF) changes in patients with retinoblastoma who have delayed treatment. METHODS: Circulating tumor DNA RB1 was detected and VAF was measured at diagnosis and again prior to any intervention at some time later ranging from 2 to 28 days. RESULTS: Four patients with five ctDNA RB1 mutations were detected at diagnosis and VAF was increased on re-evaluation of the same RB1 mutations in ctDNA. CONCLUSION: In this small cohort, every patient (4) and every RB1 mutation (5) plasma level VAF% increased when measured at two time periods before treatment was instituted suggesting that growing tumors demonstrate increasing plasma ctDNA.

Circulating Tumor DNA Posttreatment Measurements and Clinical Correlates in Retinoblastoma.

Importance: Plasma measurements of RB1 circulating tumor DNA (ctDNA) after completion of treatment may be associated with the development of metastases in patients with retinoblastoma. Objective: To determine if the absence of previously detectable plasma ctDNA is associated with metastasis-free survival in patients with a minimum of 1 year follow-up after treatment of retinoblastoma. Design, Setting, and Participants: This cohort study was conducted from June 2019 to September 2023. Patients with retinoblastoma who had measurable ctDNA levels at diagnosis and had repeated ctDNA measurements after ocular treatment (enucleation or intra-arterial chemotherapy) with a minimum of 1 year of follow-up (mean [SD], 28.2 [10.3] months) were included in the study. Patients were recruited from a single-center, tertiary cancer hospital. Exposure: Memorial Sloan Kettering's New York State-approved gene test, which interrogates 129 known cancer genes (called ACCESS), was performed on plasma samples before and after ocular treatments. All exons of the RB1 gene are included in the test and listed as ctDNA in this article. Main Outcomes and Measures: Plasma ctDNA level before treatment, after completion of ocular treatment, and development or absence of metastases. Results: A total of 24 patients (mean [SD] age, 20.7 [17.1] months; 15 female [62.5%]) were included in the study. None of the 23 patients who had a measurable ctDNA level and then no detectable ctDNA level after completion of ocular treatment developed metastases with a minimum of 1 year of follow-up. One patient had persistent measurable ctDNA after initial treatment and developed metastases. Conclusion and Relevance: Patients with retinoblastoma who had a measurable ctDNA level at diagnosis did not develop metastases if the plasma ctDNA level became unrecordable after ocular treatment; 1 patient who had persistent measurable ctDNA after treatment did develop metastasis.

How Much Time Do Focal Treatments for Retinoblastoma Add to Anesthetic Exposure?

Introduction: Children with retinoblastoma have anesthesia for exams and treatment, but there is little information about how long treatment interventions (laser, cryotherapy, and intravitreal injections) add to routine exams under anesthesia (EUA). This information would be useful for planning operating room schedules, staff schedules, family expectations, and billing. Methods: A retrospective, single-center, Institutional Review Board (IRB) approved review of anesthesia duration for retinoblastoma children undergoing EUA with laser, cryotherapy, or intravitreal injections performed at MSK between January 2019 and November 2023. Results: Three hundred eight patients had 2,399 EUAs. The average EUA lasted 24.3 min (range 7-77 min) when no interventions were done. Laser photocoagulation added an average of 18.9 min (range 19-77 min), cryotherapy 26.1 min (range 27-75 min), and intravitreal injection 23.5 min (range 10-71 min) to the basic EUA time. Bilateral laser treatments took 8 min longer than unilateral treatments. Conclusion: EUAs for children with retinoblastoma can be performed relatively quickly. Interventions such as laser, cryotherapy, or intravitreal injections roughly double the time under anesthesia but in some cases can take much longer (>1 h).

Improved method of dilating pupils for ophthalmic exams under anesthesia (faster and easier).

Objective: The pupils of children with retinoblastoma are routinely dilated pre-procedure with Tropicamide and Phenylephrine. Despite that, the pupil constricts once general anesthesia begins. The aim of this study is to see if adding Ketorolac to the regular dilating drops given pre-procedure shortens the length of anesthesia. Methods: Retrospective comparison of time under anesthesia for two groups of retinoblastoma children receiving anesthesia for examination under anesthesia: one group (January 1, 2019 to October 1, 2022) had been dilated with Tropicamide 1% and Phenylephrine 2.5% while the second group (October 2, 2022 to July 1, 2023) was dilated with a combination drop using those drugs with topical Ketorolac 0.5% and Proparacaine 0.5%. Results: Average anesthesia time for patients who received the older two-drug combination was 25 minutes vs. 16 minutes (36% reduction in exposure time) for those who received the newer four-drug combination (9 minutes less anesthesia) (P < 0.001). Conclusions: The use of a combined dilating drop that incorporated Tropicamide 1%, Phenylephrine 2.5%, Proparacaine 0.5% and Ketorolac 0.5% significantly shortened the time for exams under anesthesia for children with retinoblastoma because the pupil remained dilated after anesthesia induction with Sevoflurane. Using this combined drop, children will receive 5-10 hours less anesthesia during their treatment for retinoblastoma and staff will have more than 150 hours of fewer exposure to anesthetic gasses. In addition, far fewer drops are necessary pre-anesthesia, minimizing trauma to the children and families.

Mutant-RB1 circulating tumor DNA in the blood of unilateral retinoblastoma patients: What happens during enucleation surgery: A pilot study.

Cell free DNA (cfDNA) and circulating tumor cell free DNA (ctDNA) from blood (plasma) are increasingly being used in oncology for diagnosis, monitoring response, identifying cancer causing mutations and detecting recurrences. Circulating tumor RB1 DNA (ctDNA) is found in the blood (plasma) of retinoblastoma patients at diagnosis before instituting treatment (naïve). We investigated ctDNA in naïve unilateral patients before enucleation and during enucleation (6 patients/ 8 mutations with specimens collected 5-40 minutes from severing the optic nerve) In our cohort, following transection the optic nerve, ctDNA RB1 VAF was measurably lower than pre-enucleation levels within five minutes, 50% less within 15 minutes and 90% less by 40 minutes.

Spanish black radish (Raphanus sativus L. Var. niger) diet enhances clearance of DMBA and diminishes toxic effects on bone marrow progenitor cells.

Vegetables of the Cruciferae family contain high levels of glucosinolates, metabolites of which are believed to enhance detoxification. Spanish black radishes (SBR) contain 4× more glucosinolates than other crucifers. This study examined whether feeding mice a diet containing 20% SBR for 2 wk could enhance metabolism of 7,12-dimethylbenz(a)anthracene (DMBA) and inhibit DMBA-mediated bone marrow toxicity. Expression of Phase I and II detoxification enzymes was significantly greater for mice fed SBR than control diet. Six hours after DMBA administration, the blood levels of DMBA in mice fed the SBR diet were significantly lower than mice fed a control diet. DMBA reduced bone marrow cells in mice fed control diet to a significantly greater extent than mice fed the SBR diet. Colony forming assays demonstrated that mice on the SBR diet had 1) less reduction in lymphoid CFU-preB progenitor cells, 2) greater recovery of CFU-preB progenitor cells at 168 h, and 3) less reduction of CFU-GM progenitor cells at 6 h. Therefore, mice fed a 20% SBR diet for 2 wk had greater expression of detoxification enzymes, faster metabolism of DMBA, and a reduction in DMBA-induced bone marrow toxicity. Overall, these results support the hypothesis that glucosinolates in SBR are protective against acute toxicity.

Detectability of Plasma-Derived Circulating Tumor DNA Panel in Patients Undergoing Primary Treatment for Uveal Melanoma.

Purpose: To investigate the presence of plasma circulating tumor DNA (ctDNA) in patients with uveal melanoma during and after primary tumor treatment. Methods: Detectability and variant allele frequency of ctDNA were assessed using a 129-oncogene panel using next-generation deep sequencing and hybridization capture in 69 patients with uveal melanoma undergoing primary treatment with enucleation (n = 8, during surgery) or plaque brachytherapy (n = 61; postoperative day 0, 1, 2, or 3). Follow-up assessments were performed in 39 patients over a median of 21 months (range, 3.2-31.9 months) of follow-up. Correlations between genomic data and disease parameters were performed. Results: Overall, ctDNA was detectable in 20 of 69 patients with uveal melanoma (28.9%) during the perioperative period. On the day of enucleation, ctDNA was detected in two of eight patients (25%). In patients undergoing brachytherapy, ctDNA was significantly more detectable on postoperative days 2 or 3 compared with postoperative day 0 or 1 (32.4% vs 0.0%; P = 0.0015). Patients with follow-up ctDNA that became detectable or had an increased variant allele frequency were significantly more likely to develop metastasis compared with patients with follow-up ctDNA that became undetectable or decreased variant allele frequency (P = 0.04). In patients with detectable vs. undetectable ctDNA, there was no significant difference in tumor size, stage or location. Conclusions: ctDNA is significantly more detectable at 48 to 72 hours after plaque brachytherapy compared with less than 48 hours. ctDNA can be detected during enucleation. Relative increases in ctDNA levels may herald the development of clinically apparent metastases.

Retrospective Evaluation of Somatic Alterations in Cell-Free DNA from Blood in Retinoblastoma.

Purpose: Analysis of circulating tumor DNA (ctDNA) in the plasma of patients with retinoblastoma and simulating lesions. Design: Retrospective cross-sectional study of the association of plasma ctDNA from retinoblastoma and simulating lesions with disease course. Participants: Fifty-eight Memorial Sloan Kettering Cancer Center patients with retinoblastoma comprising 68 plasma ctDNA samples and 5 with retinoblastoma-simulating lesions. Methods: The ctDNA analyzed with hybridization capture and next-generation sequencing in blood (plasma) of patients who had retinoblastoma or simulating lesions were evaluated for association with clinical course of the disease. Main Outcome Measures: Presence or absence of molecular aberrations in the RB1 gene and correlations with clinical features. Results: RB1 cell-free DNA (cfDNA) was detected in 16 of 19 patients with newly diagnosed, untreated intraocular retinoblastoma and in 3 of 3 patients with newly diagnosed, untreated metastatic disease. It was also present in 3 patients with recurrent intraocular disease before therapy, but was not present in patients with recurrent disease who received intra-arterial chemotherapy, nor in 21 patients who had undergone enucleation for unilateral disease. In 1 patient who had delayed treatment (insurance reasons) and showed rapid growth of the intraocular tumor, the variant allele frequency increased in 1 month from 0.34% to 2.48%. No RB1 mutations were detected in the cfDNA from plasma of patients with simulating lesions (3 with Coats disease and 1 with persistent fetal vasculature [PFV]). In 2 patients, we identified 2 independent RB1 mutations in plasma. Conclusions: Mutations in RB1 were found in the cfDNA from blood of patients with newly diagnosed, untreated retinoblastoma and in patients who showed disease recurrence in the eye after prior treatment, but not in unilateral retinoblastoma after enucleation Levels of ctDNA increase in patients with progressive disease who did not receive any treatment. High plasma cfDNA levels were detected in patients with newly diagnosed metastatic disease, and these levels decreased after systemic chemotherapy was administered. Further validation is needed for measuring the somatic alterations in cfDNA from blood in retinoblastoma that could provide a promising method of monitoring patients in the future.

Aqueous extracts from dietary supplements influence the production of inflammatory cytokines in immortalized and primary T lymphocytes.

BACKGROUND: Congaplex and Immuplex are dietary supplements that have been traditionally used to support immune system function. The purpose of these experiments was to determine whether Congaplex and Immuplex affect immune function using primary and immortalized T lymphocytes. METHODS: Immortalized CEM and Jurkat T lymphocytes and primary peripheral mononuclear blood cells (PBMCs) were treated with the aqueous extracts from Congaplex and Immuplex to determine the effects of these products on cytokine production in activated T lymphocytes. RESULTS: Congaplex enhanced phytohemagglutinin/phorbol 12-myristate 13-acetate (PHA/PMA) stimulation of both CEM and Jurkat cells as measured by the production of cytokines, while Immuplex suppressed PHA/PMA-induced production of cytokines, with the exception of interleukin (IL)-8 which was enhanced by Immuplex. In vitro treatment of PBMCs from 10 healthy donors with Congaplex or Immuplex decreased PHA-stimulated production of interferon (IFN)-gamma but increased the production of IL-13. CONCLUSIONS: While the effects of Congaplex and Immuplex differed in these two models, these data demonstrate that the aqueous extracts from these two dietary supplements can affect the inflammatory response of T lymphocytes.

Heat treatment of Brussels sprouts retains their ability to induce detoxification enzyme expression in vitro and in vivo.

UNLABELLED: The bioactive metabolites of glucosinolates, such as isothiocyanates, contained in cruciferous vegetables have been shown to reduce the risk of cancers through the induction of detoxification enzymes. However, cruciferous vegetables are commonly processed before consumption, significantly altering the phytochemical composition of these vegetables. Compared to freeze-dried Brussels sprouts, oven-dried Brussels sprouts contain low concentrations of glucosinolates (22.14 and 0.85 µmol/g, respectively) and isothiocyanates (3.68 and 0.15 µmol/g, respectively). The effect of oven-dried Brussels sprouts on the expression of detoxification enzymes was evaluated in vitro and in vivo. Treatment of immortalized human hepatoma cells with the aqueous extract from oven-dried Brussels sprouts significantly increased quinone activity (0.5 and 1.5 mg/mL) and the activity of the antioxidant response element (EC50=2.39 mg/mL) and xenobiotic response element (EC50 2.92 mg/mL). C3H/HeJ mice fed a diet containing 20% oven-dried Brussels sprout diets for 2 wk demonstrated significantly higher expression than animals fed a nutrient-matched control diet of CYP1A1, CYP1A2, and epoxide hydrolase in the liver and CYP1A1, CYP1A2, CYP1B1, epoxide hydrolase, UGT1A1, thioredoxin reductase, and heme oxygenase in the lungs. The low concentrations of glucosinolates and isothiocyanates in oven-dried Brussels sprouts suggest that other compounds, such as the Maillard reaction products that are produced during heating, are responsible for the induction of detoxification enzymes in vitro and in vivo. PRACTICAL APPLICATION: The manner in which cruciferous vegetables are processed prior to consumption has significant effects on what compounds people are exposed to. The presence of glucosinolates or isothiocyanates can be a good indicator of the ability of cruciferous vegetables to induce detoxification enzymes. However, the data presented here demonstrate that while heat processing of Brussels sprouts greatly reduced the concentrations of glucosinolates and isothiocyanates, their ability to induce detoxification enzymes in vitro and in vivo was retained.

Induction of detoxification enzymes by feeding unblanched Brussels sprouts containing active myrosinase to mice for 2 wk.

In cruciferous vegetables, myrosinase metabolizes the relatively inactive glucosinolates into isothiocyanates and other products that have the ability to increase detoxification enzyme expression. Thus, maintaining myrosinase activity during food preparation may be critical to receiving the maximum benefit of consumption of Brussels sprouts or other cruciferous vegetables. To test the importance of maintaining myrosinase activity for maximizing bioactivity, experimental diets containing 20% unblanched (active myrosinase) or 20% blanched (inactivated myrosinase) freeze-dried Brussels sprouts and a nutrient-matched control diet were evaluated in vitro and in vivo for their ability to induce detoxification enzymes. Treatment of immortalized HepG2 human hepatoma cells with the unblanched Brussels sprout diet caused a greater increase quinone activity compared to the blanched Brussels sprout diet. C3H/HeJ mice fed the unblanched Brussels sprout diets for 2 wk had significantly higher plasma sulforaphane concentrations. Liver expression of CYP1A1 and epoxide hydrolase, measured using real-time PCR, was correlated with the plasma concentration of sulforaphane. In the lung, expression of epoxide hydrolase, thioredoxin reductase, UDP glucuronosyltransferase, quinone reductase, heme oxygenase, CYP1A1, CYP1A2, and CYP1B1 were also correlated with the plasma concentration of sulforaphane. Together these data demonstrate that, as predicted by the in vitro experiment, in vivo exposure to Brussels sprouts with active myrosinase resulted in greater induction of both phase I and phase II detoxification enzymes in the liver and the lungs that correlated with plasma sulforaphane concentrations.